Method for evaluating anti-aging effect of filler

Abstract

The invention provides a method for evaluating the anti-aging effect of a filling agent, and belongs to the technical field of anti-aging evaluation. According to the method, a filler is effectively injected into a corium layer of skin according to the degradation time of the filler and different injection modes, an in-vitro light aging environment is simulated by utilizing an ultraviolet irradiation model, and the content of a light aging biomarker MMP1 and the change of the content of inflammatory factors are detected by detecting the thickness of the corium layer of a skin model, the content of proCollagen I, the content of a light aging biomarker MMP1 and the content of inflammatory factors. And the anti-aging effect of the filling agent is objectively, effectively and accurately evaluated.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-aging evaluation and relates to a method for evaluating the anti-aging effect of a filler. Background Art

[0002] There are many causes of skin aging. Sun exposure, environmental pollution, long-term late sleep, smoking and alcohol abuse can affect skin health, leading to skin sagging, fine lines, coarse lines, dullness and other skin aging problems. From the perspective of skin physiology, the aging of human facial skin is mainly caused by a combination of problems such as collagen loss, cheek depression, volume loss, and soft tissue sagging.

[0003] With people's pursuit of beauty, more and more treatment methods have emerged. For example, physical injection of fillers can support facial depressions and promote collagen regeneration, which can make skin cells plump, the skin hydrated, delicate and smooth, and effectively prevent skin aging. Common cosmetic injection fillers include hyaluronic acid, collagen, poly-L-lactic acid, polycaprolactone, etc. Due to the diversity of fillers on the market, there are great differences in their relative molecular weight, degradation time, and effective time, resulting in unstable detection indicators in the detection technology and difficulty in comparing the effects of different products.

[0004] At present, the evaluation of the anti-aging effect of fillers mainly relies on biochemical experiments, 2D cell experiments or animal experiments. However, animal experiments have individual differences and are not homologous to humans; 2D cells do not conform to the actual state of human skin and cannot effectively evaluate the anti-aging effect of fillers. The 3D skin model can separate epidermal and dermal cells in a targeted manner for corresponding tests, and can locate and detect proteins at the junction of the true epidermis. At present, the sampling methods used in the 3D skin model for anti-aging efficacy testing are generally surface administration or submerged administration. Since fillers are generally large molecules, it is difficult to penetrate the epidermis, and the surface administration method prevents the filler from entering the dermis; at the same time, submerged administration will dilute the concentration of the finished filler, and fillers such as poly-L-lactic acid are not easy to mix evenly. These sampling methods make it difficult for the detection method to obtain real detection data. Summary of the invention

[0005] The purpose of the present invention is to provide a method for evaluating the anti-aging efficacy of a filler, so as to solve the problem of low accuracy of the existing evaluation methods.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present application provides a method for evaluating the anti-aging efficacy of a filler, the method comprising: S01: According to the degradation time of the filler, full-thickness skin models of a blank group, a negative control group, a sample inclusion group, a sample blending group, and a sample injection group are prepared respectively; wherein, no filler is added to the full-thickness skin models of the blank group and the negative control group, a filler with a degradation time of 21-180 days is injected into the full-thickness skin model of the sample inclusion group when collagen gel is about to be formed, a filler with a degradation time of 21-180 days is mixed into the full-thickness skin model of the sample blending group before adding the dermal fibroblast suspension; and a filler with a degradation time of 1-20 days is injected into the dermis during the air-liquid culture after the epidermal cell suspension is added to the full-thickness skin model of the sample injection group.

[0007] In this application, the full-thickness skin models of the blank group and the negative control group were prepared by the following method: S0111: Mix bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, add NaHCO3 solution, adjust the pH value to 7.2-7.4, and form a mixed solution.

[0008] S0112: The amount added to the blend is 1×10 5 -3×10 6 cells / ml of dermal fibroblast suspension, mixed evenly, added to a six-well plate transwell insert chamber, placed in a CO2 incubator and allowed to gel for 0.5-2 hours to form a collagen gel, wherein the dermal fibroblast suspension is obtained by resuspending the dermal fibroblasts in DMEM culture medium containing 10% FBS.

[0009] S0113: Add culture medium I to the collagen gel to ensure that the volume of the liquid inside the chamber is 1-3 ml and the volume of liquid inside and outside the chamber is equal. After 3-7 days of culture, discard the culture medium I and add 1×10 5 -3×10 6 Add 150 μl of epidermal cell suspension at 10 cells / ml to each well and let it stand at room temperature for 0.5-2 hours to ensure that the cells are evenly attached to the wall.

[0010] S0114: Add culture medium II to the inside and outside of the chamber to ensure the balance of the liquid level inside and outside the chamber, and place it in a CO2 incubator for 2-7 days. After the culture is completed, discard the culture medium II, add the keratinization medium culture medium, and culture it at the gas-liquid surface for 7-14 days, changing the liquid every day to form a full-thickness skin model.

[0011] The method for preparing the full-thickness skin model of the sample group in this application includes: S0121: Mix bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, add NaHCO3 solution, adjust the pH value to 7.2-7.4, and form a mixed solution.

[0012] S0122: The amount added to the blend is 1×10 5 -3×10 6 cells / ml of dermal fibroblast suspension, and mixed evenly to form a dermal fibroblast mixed solution.

[0013] S0123: Add 2 / 3 volume of dermal fibroblast mixture to the six-well plate transwell insert chamber, and place it in a CO2 incubator to allow gelation for 0.5-1h. When the collagen gel is about to form, inject 100μl of filler with a long degradation time using a syringe, add the remaining 1 / 3 volume of dermal fibroblast mixture to seal the needle hole, and continue to place it in a CO2 incubator to allow gelation for 0.3-1h to form a collagen gel; wherein, the dermal fibroblast suspension is obtained by resuspending the dermal fibroblasts in DMEM culture medium containing 10% FBS.

[0014] S0124: Add culture medium I to the collagen gel to ensure that the volume of the liquid inside the chamber is 1-3 ml and the volume of liquid inside and outside the chamber is equal. After 3-7 days of culture, discard the culture medium I and add 1×10 5 -3×10 6 Add 150 μl of epidermal cell suspension at 10 cells / ml to each well and let it stand at room temperature for 0.5-2 hours to ensure that the cells are evenly attached to the wall.

[0015] S0125: Add culture medium II to the inside and outside of the chamber to ensure the balance of the liquid level inside and outside the chamber, and place it in a CO2 incubator for 2-7 days. After the culture is completed, discard the culture medium II, add the keratinization medium culture medium, and culture it at the gas-liquid surface for 7-14 days, changing the liquid every day to form a full-thickness skin model.

[0016] The preparation method of the full-thickness skin model of the sample blending group in this application includes: S0131: After mixing bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, NaHCO3 solution was added and the pH value was adjusted to 7.2-7.4 to form a mixed solution; S0132: Add 100 μl of filler with long degradation time to the blended liquid, mix well, and add 1×10 5 -3×10 6 cells / ml of dermal fibroblast suspension, mixed evenly, added to a six-well plate transwell insert chamber, placed in a CO2 incubator and allowed to gel for 0.5-2 hours to form a collagen gel; wherein the dermal fibroblast suspension is obtained by resuspending the dermal fibroblasts in DMEM culture medium containing 10% FBS.

[0017] S0133: Add culture medium I to the collagen gel to ensure that the volume of the liquid inside the chamber is 1-3 ml and the volume of liquid inside and outside the chamber is equal. After 3-7 days of culture, discard the culture medium I and add 1×10 5 -3×10 6 Add 150 μl of epidermal cell suspension at 10 cells / ml to each well and let it stand at room temperature for 0.5-2 hours to ensure that the cells are evenly attached to the wall.

[0018] S0134: Add culture medium II to the inside and outside of the chamber to ensure the balance of the liquid level inside and outside the chamber, and place it in a CO2 incubator for 2-7 days. After the culture is completed, discard the culture medium II, add the keratinization medium culture medium, and culture it at the gas-liquid surface for 7-14 days, changing the liquid every day to form a full-thickness skin model.

[0019] In this application, the preparation method of the full-thickness skin model of the sample injection group includes: S0141: Mix bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, add NaHCO3 solution, adjust the pH value to 7.2-7.4, and form a mixed solution.

[0020] S0142: The amount added to the blend is 1×10 5 -3×10 6 cells / ml of dermal fibroblast suspension, mixed evenly, added to a six-well plate transwell insert chamber, placed in a CO2 incubator and allowed to gel for 0.5-2 hours to form a collagen gel; wherein the dermal fibroblast suspension is obtained by resuspending the dermal fibroblasts in DMEM culture medium containing 10% FBS.

[0021] S0143: Add culture medium I to the collagen gel to ensure that the volume of the liquid inside the chamber is 1-3 ml and the volume of liquid inside and outside the chamber is equal. After 3-7 days of culture, discard the culture medium I and add 1×10 5 -3×10 6 Add 150 μl of epidermal cell suspension at 10 cells / ml to each well and let it stand at room temperature for 0.5-2 hours to ensure that the cells are evenly attached to the wall.

[0022] S0144: Add culture medium II to the inside and outside of the chamber to ensure the balance of the liquid level inside and outside the chamber, and place it in a CO2 incubator for 2-7 days. After the culture is completed, discard the culture medium II, add the keratinization medium culture medium, and culture the air-liquid surface for 7-14 days, changing the liquid every day to form a full-thickness skin model. On the 1st to 14th day of the air-liquid culture, use a syringe to inject 100μl of a filler with a short degradation time into the dermis. Among them, the injection site of the filler is 1 point on the full-thickness skin model, or 4 intersections of 2 horizontal and 2 vertical, or 6 intersections of 2 horizontal and 3 vertical, or 9 intersections of 3 horizontal and 3 vertical, etc.

[0023] In the present application, a long degradation time refers to a degradation time of 21-180 days, and a short degradation time refers to a degradation time of 1-20 days.

[0024] During the preparation of the full-thickness skin models of the blank group, negative control group, sample inclusion group, sample blending group, and sample injection group, culture medium I was DMEM culture medium containing 10% FBS, and the culture medium included 50-100ug / ml vitamin C; culture medium II was 70% DMEM, 30% F12, 0.1-10% FBS, hydrocortisone 0.2-0.8μg / ml, insulin 1-50μg / ml, adenine 10-50μg / ml, selenium 0.05-2nM, progesterone 0.1-3nM, transferrin 10-20ng / ml, and epidermal growth factor 0.2-10ng / ml.

[0025] S02: performing UVR irradiation on the full-thickness skin models of the negative control group, the sample inclusion group, the sample blending group, and the sample injection group, and incubating the blank group and the irradiated full-thickness skin models.

[0026] Specifically, the culture medium in the full-thickness skin model of the blank group, negative control group, sample inclusion group, sample blending group, and sample injection group was discarded, and 1 ml of PBS (phosphate buffered saline) was added to each well. The blank group was protected from light, and the remaining components were first irradiated with UVA and then with UVB, where the dose of UVA irradiation was 10-50 J / cm 2 The irradiation time is 1000s-5000s; the UVB irradiation dose is 50-600mJ / cm 2 The irradiation time is 50s-600s; the total irradiation times are 1-5 times, once a day.

[0027] Incubate the blank and irradiated full-thickness skin models for 1-3 days.

[0028] S03: Detect the dermis thickness, proCollagen I content, photoaging biomarker MMP1 content, and inflammatory factor content of the full-thickness skin model after incubation.

[0029] Samples were taken from the full-thickness skin models after incubation, and the dermis thickness, proCollagen I content, photoaging biomarker MMP1 content, and inflammatory factor content of the full-thickness skin models in each group were detected.

[0030] Among them, the dermis thickness detection is to take the full-thickness skin model after incubation, embed it in paraffin and slice it, and then perform HE staining to observe and measure the dermis thickness through the staining picture. The proCollagen I content detection is to take the full-thickness skin model after incubation, embed it in paraffin and slice it, and then perform immunofluorescence staining, and use Image J to analyze the fluorescent picture to obtain the proCollagen I content. The photoaging biomarker MMP1 content detection is to take the full-thickness skin model after incubation, embed it in paraffin and slice it, and then perform immunohistochemistry, and use Image J to analyze the stained picture to obtain the MMP1 content. The inflammatory factor content detection is to recover the culture medium after the incubation is completed, and use the ELISA kit to detect the inflammatory factor content in the full-thickness skin model, wherein the inflammatory factors include one or more of IL-1α, IL-6, IL-8 and TNF-α.

[0031] S04: Relative to the full-thickness skin model of the negative control group, if the full-thickness skin model of the sample inclusion group, the sample blending group, or the sample injection group satisfies any one or more of increased dermis thickness, increased proCollagen I content, decreased photoaging biomarker MMP1 content, or decreased inflammatory factor content, then the filler in the full-thickness skin model of the sample inclusion group, the sample blending group, or the sample injection group has anti-aging efficacy.

[0032] After the dermis thickness, proCollagen I content, photoaging biomarker MMP1 content, and inflammatory factor content were detected, GraphPad Prism was used to draw graphs, and the results were expressed as (mean ± standard deviation). The t-test was used for statistical analysis among the groups, and * P < 0.05, ** P < 0.01, # P<0.05, ## P<0.01 indicates that the difference is statistically significant. Among them, # indicates the significant analysis results of the control group and the blank group; * indicates the significant analysis results of the sample group and the control group.

[0033] It was determined through the GraphPad Prism graph that, relative to the full-thickness skin model of the negative control group, if the full-thickness skin model of the sample inclusion group, the sample blending group, and the sample injection group met any one or more of the following conditions: the thickness of the dermis was significantly increased with a statistically significant difference; the content of proCollagen I was significantly increased with a statistically significant difference, the content of the photoaging biomarker MMP1 was significantly decreased with a statistically significant difference, or the content of inflammatory factors was significantly decreased with a statistically significant difference, then the filler in the full-thickness skin model of the sample inclusion group, the sample blending group, and the sample injection group had anti-aging efficacy.

[0034] The present invention has the following beneficial effects: (1) Considering that fillers basically act on the dermis of the skin, this application adopts different sampling methods such as inclusion method, blending method or injection method to place the filler in the dermis of the skin model to test the anti-aging effect of the filler. Since the filler acts directly on the dermis, the utilization rate of the filler is guaranteed, and the test results are accurate and true.

[0035] (2) In the present application, the inclusion method is to inject the filler into the collagen gel that is about to gel, which can simulate the single-point injection method; the blending method is to blend the filler with the collagen gel of the full-length skin model, which can simulate the multi-point continuous injection method such as a nine-needle syringe.

[0036] (3) In the present application, different sampling methods are selected according to the degradation time of the filler, and then different anti-aging efficacy evaluation methods are determined. Among them, the filler with a short degradation time is sampled during the gas-liquid culture period, and the filler with a long degradation time is sampled by the blending method or the inclusion method. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a comparison chart of the dermis thickness of the full-thickness skin models of each group in the embodiment; Figure 2 It is a comparison chart of the proCollagen I content of the full-thickness skin models of each group in the example; Figure 3 This is a comparison chart of the photoaging biomarker MMP1 content in the full-thickness skin models of each group in the example; Figure 4 This is a comparison chart of the inflammatory factor IL-6 content in the full-thickness skin models of each group in the example; Figure 5 This is a comparison chart of the inflammatory factor IL-8 content in the full-thickness skin models of each group in the example; Figure 6 It is a comparison chart of the dermis thickness of the full-thickness skin models in each group of comparative examples; Figure 7 This is a comparison chart of the proCollagen I content in the full-thickness skin models of each group of comparative examples; Figure 8 This is a comparison chart of the photoaging biomarker MMP1 content in the full-thickness skin models of each group of comparative examples; Fig. 9 This is a comparison chart of the inflammatory factor IL-8 content in the full-thickness skin models of each group in the comparative example. DETAILED DESCRIPTION

[0038] In the embodiments of the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry, and the methods in the following embodiments, unless otherwise specified, are conventional methods in the art. The technical solution of the present invention is further explained and illustrated by specific embodiments.

[0039] Example The present application provides a method for evaluating the anti-aging efficacy of a filler, wherein the filler is hyaluronic acid, and the method comprises: S101: Prepare full-thickness skin models of blank group, negative control group, sample inclusion group, sample blending group, and sample injection group respectively.

[0040] (1) The preparation method of the full-thickness skin model of the blank group and the negative control group includes: After mixing bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, NaHCO3 solution was added and the pH value was adjusted to 7.2 to form a mixed solution. 1×10 5 The dermal fibroblast suspension of 100 cells / ml was mixed evenly and added to the transwell insert chamber of the six-well plate. The chamber was placed in a CO2 incubator and allowed to gel for 2 hours to form a collagen gel. The dermal fibroblast suspension was obtained by resuspending the dermal fibroblasts in DMEM medium containing 10% FBS. Culture medium I was added to the collagen gel to ensure that the volume of the liquid in the chamber was 3 ml and the volume of the liquid inside and outside the chamber was equal. After 5 days of culture, the culture medium I was discarded and 1×10 5 Add 150 μl of epidermal cell suspension of 10 cells / ml to each well and let it stand at room temperature for 2 hours to ensure that the cells are evenly attached to the wall. Add culture medium II to the inside and outside of the chamber to ensure the balance of the liquid level inside and outside the chamber, and place it in a CO2 incubator for 5 days. After the culture is completed, discard the culture medium II and add the keratinization medium culture medium to culture at the air-liquid surface for 12 days, changing the liquid every day to form a full-thickness skin model.

[0041] (2) The method for preparing the full-thickness skin model of the sample inclusion group includes: After mixing bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, NaHCO3 solution was added and the pH value was adjusted to 7.2 to form a mixed solution. 1×10 5 -3×10 6cells / ml of dermal fibroblast suspension, mix evenly to form a dermal fibroblast blend. Add 2 / 3 volume of dermal fibroblast blend to the six-well plate transwell insert chamber, mix evenly, and place in a CO2 incubator to gel for 1 hour. When the collagen gel is about to form, inject 100μl of hyaluronic acid filler with a syringe, add the remaining 1 / 3 volume of dermal fibroblast blend to seal the pinhole, and continue to place in a CO2 incubator to gel for 1 hour to form a collagen gel; wherein, the dermal fibroblast suspension is obtained by resuspending the dermal fibroblasts in DMEM medium containing 10% FBS, and the amount of dermal fibroblasts used is 1×10 5 cells / ml. Add culture medium I to the collagen gel to ensure that the volume of the liquid inside the chamber is 3 ml and the volume of liquid inside and outside the chamber is equal. After 5 days of culture, discard the culture medium I and add 1×10 5 Add 150 μl of epidermal cell suspension of 10 cells / ml to each well and let it stand at room temperature for 2 hours to ensure that the cells are evenly attached to the wall. Add culture medium II to the inside and outside of the chamber to ensure the balance of the liquid level inside and outside the chamber, and place it in a CO2 incubator for 5 days. After the culture is completed, discard the culture medium II and add the keratinization medium culture medium to culture at the air-liquid surface for 12 days, changing the liquid every day to form a full-thickness skin model.

[0042] (3) The preparation method of the full-thickness skin model of the sample blending group includes: Bovine type I collagen, newborn calf serum and 10×DEME were mixed in a volume ratio of 8:1:1, and NaHCO3 solution was added to adjust the pH value to 7.2 to form a mixed solution; 100 μl of hyaluronic acid filler was added to the mixed solution, mixed evenly, and then 1×10 5 cells / ml of dermal fibroblast suspension, mixed evenly, added to the six-well plate transwell insert chamber, placed in a CO2 incubator to gel for 2 hours to form collagen gel; wherein, the dermal fibroblast suspension is obtained by resuspending dermal fibroblasts in DMEM medium containing 10% FBS. Add culture medium I to the collagen gel to ensure that the volume of the liquid in the chamber is 3 ml and the volume of the liquid inside and outside the chamber is equal. After 5 days of culture, discard culture medium I and add 1×10 5 Add 150 μl of epidermal cell suspension of 10 cells / ml to each well and let it stand at room temperature for 2 hours to ensure that the cells are evenly attached to the wall. Add culture medium II to the inside and outside of the chamber to ensure the balance of the liquid level inside and outside the chamber, and place it in a CO2 incubator for 5 days. After the culture is completed, discard the culture medium II and add the keratinization medium culture medium to culture at the air-liquid surface for 12 days, changing the liquid every day to form a full-thickness skin model.

[0043] (4) The preparation method of the full-thickness skin model of the sample injection group includes: After mixing bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, NaHCO3 solution was added and the pH value was adjusted to 7.2 to form a mixed solution. 1×10 5 cells / ml of dermal fibroblast suspension, mixed evenly, added to the six-well plate transwell insert chamber, placed in a CO2 incubator to gel for 2 hours to form collagen gel; wherein, the dermal fibroblast suspension is obtained by resuspending dermal fibroblasts in DMEM medium containing 10% FBS. Add culture medium I to the collagen gel to ensure that the liquid volume in the chamber is 3ml and the liquid volume inside and outside the chamber is equal. After 5 days of culture, discard culture medium I and add 1×10 5 Cells / ml epidermal cell suspension, add 150μl to each well, let stand at room temperature for 2h to ensure that the cells are evenly attached. Add culture medium II to the inside and outside of the chamber to ensure the balance of the liquid level inside and outside the chamber, and place it in a CO2 incubator for 5 days. After the culture is completed, discard the culture medium II, add the keratinization medium culture medium, and culture it at the air-liquid surface for 12 days, changing the liquid every day to form a full-thickness skin model. The injection sampling group uses the injection sampling method to inject the sample into the dermis during the air-liquid period. The injection site is the 9 intersections of 3 horizontal and 3 vertical on the dermis.

[0044] S102: The culture medium in the full-thickness skin model of the blank group, negative control group, sample inclusion group, sample blending group, and sample injection group was discarded, and 1 ml of PBS was added to each well. The blank group was protected from light, and the other groups were first irradiated with UVA and then with UVB, where the dose of UVA irradiation was 30 J / cm 2 The irradiation time is 3000s / time; the UVB irradiation dose is 100 mJ / cm 2 The irradiation time was 100s / time; the total number of irradiations was 3 times, once a day. After the last irradiation, the blank group and the irradiated full-thickness skin model were incubated for 1 day. The injection time of the sample group was after the first irradiation, and the sample was injected once.

[0045] S103: Sampling was performed from the full-thickness skin model after incubation to detect the dermis thickness, proCollagen I content, photoaging biomarker MMP1 content, and inflammatory factor content of the full-thickness skin model of each group.

[0046] The cut samples were fixed with 4% paraformaldehyde for 24 hours, then dehydrated with ethanol gradient, immersed in pure toluene, embedded in paraffin, and sliced. The skin sections were stained with HE (hematoxylin and eosin) staining kit to obtain the attached Figure 1 .

[0047] The cut samples were fixed with 4% paraformaldehyde. After fixation for 24 hours, they were sequentially dehydrated with ethanol gradient, immersed in pure toluene, embedded in paraffin, sliced, dewaxed to water, antigen repaired, serum blocked, incubated with primary antibody, incubated with secondary antibody, counterstained with DAPI for cell nuclei, quenched tissue autofluorescence, and sealed. At least 3 images were taken for each full-thickness skin model sealing sample, and the grayscale analysis of the green fluorescence image was performed using the image processing software Image J. The average fluorescence intensity of the image was calculated to obtain the content of proCollagen I. The proCollagen I content of the full-thickness skin model in each group is shown in the attached figure. Figure 2 shown.

[0048] The cut samples were fixed with 4% paraformaldehyde. After fixation for 24 hours, they were sequentially dehydrated with ethanol gradient, immersed in pure toluene, embedded in paraffin, sliced, dewaxed and treated with water. The sections after antigen heat repair were incubated in 5% H2O2 to eliminate the activity of endogenous peroxidase, and PBS containing 4% BSA was added to block its nonspecific binding. The sections were incubated with MMP-1 antibody at room temperature overnight, incubated with secondary antibody, and counterstained with DAPI. The sections were photographed under a microscope, and the images were collected and analyzed using Image J image processing software to obtain the attached Figure 3 .

[0049] After the full-thickness skin model incubation was completed, the culture medium was collected and the inflammatory factors IL-6 and IL-8 were detected using an ELISA kit. The data software was selected to calculate the standard curve, and the OD value of the sample to be tested was substituted into the standard curve to calculate the IL-6 and IL-8 content. Figure 4 , 5 .

[0050] S104: By the attached Figure 1 It can be seen that compared with the negative control group, the thickness of the dermis in the sample inclusion method, sample blending group and sample injection group increased significantly, and there was a statistically significant difference. Figure 2 It can be seen that compared with the negative control group, the proCollagen I content in the sample inclusion method, sample blending group and sample injection group increased significantly, and there was a statistically significant difference. Figure 3 It can be seen that compared with the negative control group, the levels of photoaging biomarker MMP1 in the sample inclusion method, sample blending group and sample injection group were significantly decreased, and there was a statistically significant difference. Figure 4 , 5It can be seen that compared with the negative control group, the levels of inflammatory factors IL-6 and IL-8 in the sample inclusion method, sample blending group and sample injection group were significantly reduced, and there was a statistically significant difference. The sample inclusion method, sample blending group and sample injection group all met the above conditions, which can indicate that hyaluronic acid fillers have anti-aging effects.

[0051] Comparative Example 1 The comparative example of the present application provides a method for evaluating the anti-aging efficacy of a filler by a surface spreading method, wherein the filler is collagen, and the method comprises: D101: The preparation method of the full-thickness skin model of the blank group, negative control group and surface application group is as follows: bovine type I collagen, newborn calf serum and 10×DEME were mixed at a volume ratio of 8:1:1, and then NaHCO3 solution was added to adjust the pH value to 7.2 to form a mixed solution. 1×10 5 The dermal fibroblast suspension of 100 cells / ml was mixed evenly and added to the transwell insert chamber of the six-well plate. The chamber was placed in a CO2 incubator and allowed to gel for 2 hours to form a collagen gel. The dermal fibroblast suspension was obtained by resuspending the dermal fibroblasts in DMEM medium containing 10% FBS. Culture medium I was added to the collagen gel to ensure that the volume of the liquid in the chamber was 3 ml and the volume of the liquid inside and outside the chamber was equal. After 5 days of culture, the culture medium I was discarded and 1×10 5 Add 150 μl of epidermal cell suspension of 10 cells / ml to each well and let it stand at room temperature for 2 hours to ensure that the cells are evenly attached to the wall. Add culture medium II to the inside and outside of the chamber to ensure the balance of the liquid level inside and outside the chamber, and place it in a CO2 incubator for 5 days. After the culture is completed, discard the culture medium II and add the keratinization medium culture medium to culture at the air-liquid surface for 12 days, changing the liquid every day to form a full-thickness skin model.

[0052] D102: The culture medium in the full-thickness skin model of the blank group, negative control group and surface application group was discarded, and 1 ml of PBS was added to each well. The blank group was protected from light, and the other groups were first irradiated with UVA and then with UVB. The dose of UVA irradiation was 25 J / cm 2 The irradiation time was 2500s; the UVB irradiation dose was 100mJ / cm 2 , irradiation time was 100s; irradiation was performed once. After the first irradiation, the surface smearing group was sampled by surface smearing, and 100μl collagen filler was applied to each well. After incubation for 24h, irradiation was performed twice continuously, and 1ml of culture medium was added after each irradiation, and incubation continued for 24h.

[0053] D103: Sampling was performed from the full-thickness skin model after incubation to detect the dermis thickness, proCollagen I content, photoaging biomarker MMP1 content, and inflammatory factor content of each group of full-thickness skin models. The detection process was the same as in Example 1. Figure 6-9 .

[0054] D104: By the attached Figure 6-9 It can be seen that compared with the negative control group, the dermis thickness, proCollagen I content, photoaging biomarker MMP1 content, and inflammatory factor IL-8 content of the surface application group did not change much, and there was no significant difference, which shows that surface application samples cannot be used to evaluate the anti-aging effect of collagen fillers. This may be because the molecular weight of collagen fillers is large and cannot penetrate the epidermis of the model to reach the dermis of the skin to work.

[0055] From the above content, it can be seen that the inclusion method, blending method and injection method can all be used to evaluate the anti-aging effect of fillers, while the surface addition method is not suitable for evaluating the anti-aging effect of fillers, that is, the surface addition method cannot make the filler have anti-aging effect.

[0056] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for evaluating the anti-aging efficacy of a filler, characterized in that: include: According to the degradation time of the filler, full-thickness skin models of a blank group, a negative control group, a sample inclusion group, a sample blending group, and a sample injection group were prepared respectively; wherein, no filler was added to the full-thickness skin models of the blank group and the negative control group, a filler with a degradation time of 21-180 days was injected into the full-thickness skin model of the sample inclusion group when the collagen gel was about to be formed, and a filler with a degradation time of 21-180 days was mixed into the full-thickness skin model of the sample blending group before adding the dermal fibroblast suspension; and a filler with a degradation time of 1-20 days was injected into the dermis during the air-liquid culture after adding the epidermal cell suspension into the full-thickness skin model of the sample injection group; Performing UVR irradiation on the full-thickness skin models of the negative control group, the sample inclusion group, the sample blending group, and the sample injection group, and incubating the blank group and the irradiated full-thickness skin models; Detecting the dermis thickness, proCollagen I content, photoaging biomarker MMP1 content, and inflammatory factor content of the full-thickness skin model after incubation; wherein the inflammatory factor is one or more of IL-1α, IL-6, IL-8, and TNF-α; Relative to the full-thickness skin model of the negative control group, if the full-thickness skin model of the sample inclusion group, the sample blending group, or the sample injection group satisfies any one or more of an increase in dermal thickness, an increase in proCollagen I content, a decrease in the photoaging biomarker MMP1 content, or a decrease in the inflammatory factor content, then the filler in the full-thickness skin model of the sample inclusion group, the sample blending group, or the sample injection group has anti-aging efficacy.

2. The method for evaluating the anti-aging efficacy of fillers according to claim 1, characterized in that: UVR irradiation includes UVA irradiation first, and then UVB irradiation, where the dose of UVA irradiation is 10-50 J / cm 2 The irradiation time is 1000s-5000s; the UVB irradiation dose is 50-600mJ / cm 2 The irradiation time is 50s-600s; the total irradiation times are 1-5 times, once a day.

3. The method for evaluating the anti-aging efficacy of fillers according to claim 1, characterized in that: The full-thickness skin models of the blank group and the negative control group were prepared by the following method: After mixing bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, NaHCO3 solution was added and the pH value was adjusted to 7.2-7.4 to form a mixed solution; Add the dermal fibroblast suspension to the blended solution, mix well, and let the gel stand for 0.5-2 hours to form a collagen gel; After adding culture medium I to the collagen gel for 3-7 days, discarding culture medium I, adding epidermal cell suspension, and standing at room temperature for 0.5-2 hours; After adding culture medium II for 2-7 days of culture, medium II was discarded and keratinization medium culture medium was added for air-liquid surface culture for 7-14 days, with the medium changed every day to form a full-thickness skin model.

4. The method for evaluating the anti-aging efficacy of fillers according to claim 1, characterized in that: The method for preparing the full-thickness skin model of the sample-containing group comprises: After mixing bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, NaHCO3 solution was added and the pH value was adjusted to 7.2-7.4 to form a mixed solution; Adding the dermal fibroblast suspension into the mixed solution and mixing evenly to form a dermal fibroblast mixed solution; Take 2 / 3 volume of dermal fibroblast mixture and let it stand for 0.5-1h to gel; when collagen gel is about to form, inject filler with a syringe, add 1 / 3 volume of dermal fibroblast mixture on the surface of collagen gel, let it stand for 0.3-1h to form collagen gel; After adding culture medium I to the collagen gel for 3-7 days, discarding culture medium I, adding epidermal cell suspension, and standing at room temperature for 0.5-2 hours; After adding culture medium II for 2-7 days of culture, medium II was discarded and keratinization medium culture medium was added for air-liquid surface culture for 7-14 days, with the medium changed every day to form a full-thickness skin model.

5. The method for evaluating the anti-aging efficacy of fillers according to claim 1, characterized in that: The preparation method of the full-thickness skin model of the sample blending group comprises: After mixing bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, NaHCO3 solution was added and the pH value was adjusted to 7.2-7.4 to form a mixed solution; Add the filler to the blended liquid, mix well, add the dermal fibroblast suspension, mix well, and let the gel stand for 0.5-2 hours to form a collagen gel; After adding culture medium I to the collagen gel for 3-7 days, discarding culture medium I, adding epidermal cell suspension, and standing at room temperature for 0.5-2 hours; After adding culture medium II for 2-7 days of culture, medium II was discarded and keratinization medium culture medium was added for air-liquid surface culture for 7-14 days, with the medium changed every day to form a full-thickness skin model.

6. The method for evaluating the anti-aging efficacy of fillers according to claim 1, characterized in that: The preparation method of the full-thickness skin model of the sample injection group comprises: After mixing bovine type I collagen, newborn calf serum and 10×DEME in a volume ratio of 8:1:1, NaHCO3 solution was added and the pH value was adjusted to 7.2-7.4 to form a mixed solution; Add the dermal fibroblast suspension to the blended solution, mix well, and let the gel stand for 0.5-2 hours to form a collagen gel; After adding culture medium I to the collagen gel for 3-7 days, discarding culture medium I, adding epidermal cell suspension, and standing at room temperature for 0.5-2 hours; After adding culture medium II and culturing for 2-7 days, culture medium II is discarded, and keratinization medium culture medium is added, and air-liquid surface culture is carried out for 7-14 days, and the medium is changed every day to form a full-thickness skin model; during the air-liquid culture process, the filler is injected into the dermis with a syringe; wherein, the injection site is 1 point on the full-thickness skin model, or 4 intersections of 2 horizontal and 2 vertical, or 6 intersections of 2 horizontal and 3 vertical, or 9 intersections of 3 horizontal and 3 vertical.

7. The method for evaluating the anti-aging efficacy of a filler according to any one of claims 3 to 6, characterized in that: The dermal fibroblast suspension is obtained by resuspending dermal fibroblasts in a DMEM culture medium containing 10% FBS.

8. The method for evaluating the anti-aging efficacy of a filler according to any one of claims 3 to 6, characterized in that: The amount of dermal fibroblasts in the dermal fibroblast suspension is 1×10 5 -3×10 6 cells / ml.

9. The method for evaluating the anti-aging efficacy of a filler according to any one of claims 3 to 6, characterized in that: The amount of epidermal cells in the epidermal cell suspension is 1×10 5 -3×10 6 cells / ml.

10. The method for evaluating the anti-aging efficacy of a filler according to any one of claims 3 to 6, characterized in that: The culture medium I is a DMEM culture medium containing 10% FBS, and the culture medium includes 50-100ug / ml vitamin C; the culture medium II includes 70% DMEM, 30% F12, 0.1-10% FBS, 0.2-0.8μg / ml hydrocortisone, 1-50μg / ml insulin, 10-50μg / ml adenine, 0.05-2nM selenium, 0.1-3nM progesterone, 10-20ng / ml transferrin and 0.2-10ng / ml epidermal growth factor.

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